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Originally published In Press as doi:10.1074/jbc.M607010200 on September 7, 2006

J. Biol. Chem., Vol. 281, Issue 45, 33971-33981, November 10, 2006
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Smad4-dependent Regulation of Urokinase Plasminogen Activator Secretion and RNA Stability Associated with Invasiveness by Autocrine and Paracrine Transforming Growth Factor-beta*

Sheng-Ru Shiou{ddagger}, Pran K. Datta§||, Punita Dhawan, Brian K. Law**, Jonathan M. Yingling{ddagger}{ddagger}, Dan A. Dixon¶¶, and R. Daniel Beauchamp{ddagger}§||1

From the Departments of {ddagger}Cell and Developmental Biology, §Cancer Biology, and Surgery and the ||Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2730, the **Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32611-9500, {ddagger}{ddagger}Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, and the ¶¶Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208-0002

Received for publication, July 24, 2006 , and in revised form, September 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant tumors are characterized by their ability to metastasize to distant organs. The initial steps of metastasis involve invasive growth of tumors across the basement membrane and migration through the extracellular matrix (ECM).2 Because the enzymatic degradation of both the basement membrane and ECM barriers requires a number of ECM-degrading proteases (1, 2) and is a critical early event in metastasis, invasiveness may be modulated by the expression of ECM-degrading proteases in tumor cells in response to autonomous and microenvironmental signals. Among the increasing number of ECM-degrading proteases implicated in metastasis, considerable attention has been focused on the family of matrix metalloproteinases (MMPs) and the plasminogen activator system. One of the regulators of these ECM-degrading proteases is transforming growth factor-beta (TGF-beta).

TGF-beta is a multifunctional cytokine that regulates cell proliferation, differentiation, plasticity, and migration in a context-dependent manner (reviewed in Refs. 3 and 4). TGF-beta transduces signaling through a transmembrane type II receptor (TbetaRII), a constitutively active serine/threonine kinase receptor (5). Upon ligand binding, the TbetaRII recruits and transphosphorylates intracellular TGF-beta type I receptor (TbetaRI), thereby stimulating TbetaRI serine/threonine kinase activity (6). The TbetaRI then activates the downstream effectors, Smad2 and Smad3, by phosphorylation. The activated Smad proteins form complexes with the common Smad mediator, Smad4, and then translocate to the nucleus, where the Smad complexes regulate transcription of TGF-beta target genes in conjunction with various transcriptional or co-transcriptional regulators. In addition to the Smad pathway, other signaling pathways, including the extracellular signal-regulated kinases (ERK1/2) (7, 8), the mitogen-activated protein kinase (p38) (9, 10), the Src (11), and the phosphatidylinositol 3-kinase (PI3K) (12) pathways can be activated by TGF-beta in a context-dependent manner. The precise molecular mechanisms of regulation of these pathways for TGF-beta signaling and the physiological and pathological roles of TGF-beta in normal tissues and cancer have not been completely defined.

The importance of autocrine TGF-beta in tumor progression and metastatic behavior has been documented previously. For instance, disruption of autocrine TGF-beta signaling by a dominant-negative type II receptor (DNIIR) inhibited the invasive and metastatic potential of mammary and colon carcinoma cells (13). This was attributed to prevention of autocrine TGF-beta-induced epithelial-to-mesenchymal transition, a process believed to promote tumor cell migration and invasion (12). In a different study, overexpression of a soluble TGF-beta type III receptor antagonized autocrine TGF-beta activity and resulted in inhibition of tumor cell proliferation and induction of apoptosis (14).

The urokinase plasminogen activator (uPA) is a serine protease capable of initiating cascades of activation of ECM-degrading enzymes (15) and eliciting intracellular signaling through receptor binding. Clinically, elevated uPA expression in tumors is associated with tumor aggressiveness and poor outcome in patients (16, 17) and numerous studies have linked uPA to invasive and metastatic phenotype of tumors in vitro and in animal models (18–21). The metastatic MDA-MB-231 breast cancer cells secrete active TGF-beta (22, 23) and are TGF-beta-responsive (24). These cells also express both the matrix metalloproteinases-9 (MMP-9) and uPA (20, 25, 26). We hypothesized that autocrine TGF-beta may function as a tumor promoter by regulating MMP-9 or uPA activity in MDA-MB-231 cells. The present study provides evidence that autocrine TGF-beta regulates both cell invasiveness and uPA secretion. Inhibition of uPA activity is sufficient to suppress tumor cell invasion to the same extent as inhibition of autocrine TGF-beta signaling, suggesting that autocrine TGF-beta stimulation of invasiveness occurs via its regulation of uPA release. The Smad pathway appears to be required for the regulation of uPA release as silencing of Smad4 protein expression suppressed uPA secretion. Interestingly, although autocrine TGF-beta regulates uPA production through protein secretion, exogenous TGF-beta further increases uPA expression through RNA stabilization also through a Smad4-dependent fashion. Finally, this work demonstrates that a pharmacological kinase inhibitor of TGF-beta receptors inhibits both uPA secretion and tumor cell invasiveness, thereby providing evidence for the potential efficacy of targeting TGF-beta signaling for therapeutic intervention in cancer and suggests that uPA expression or secretion may be an important mediator of such effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Reagents—MDA-MB-231 cells from the American Type Culture Collection (Manassas, VA) were maintained in the Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 37 °C incubator with 5% CO2. TGF-beta was purchased from R & D Systems (Minneapolis, MN). The recombinant active human PAI-1 (cat. no. 1092) and human urokinase (cat. no. 124) were from American Diagnostica, Inc. (Greenwich, CT). The pharmacological inhibitor of the TGF-beta type I receptor (LY364947) (27, 28) was provided by Eli Lilly (Indianapolis, IN).

Immunoblot Analysis—To harvest protein lysates, cells were washed with cold phosphate-buffered saline (PBS) and lysed in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) for 20 min on ice. Lysates were sonicated and then clarified by centrifugation at 15,000 x g for 15 min at 4 °C. Protein contents of lysates were determined by the Bradford Assay (Bio-Rad). Proteins in the lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% milk PBS-T (0.1% Tween 20 (v/v) in PBS) for 1 h at room temperature and then probed with primary antibodies in 5% milk PBS-T overnight at 4 °C. After several washes with PBS-T, membranes were incubated in PBS-T containing horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and washed again with PBS-T. Immunoreactive bands were visualized by chemiluminescence reaction using ECL reagents (Amersham Biosciences) followed by exposure of the membranes to XAR5 films (Kodak, Rochester, NY). To detect secreted uPA, conditioned media were collected, centrifuged at 15,000 x g for 5 min to remove cell debris, and then subjected to immunoblotting under non-reducing conditions. 2- to 8-h conditioned media were concentrated using Microcon YM-10 centrifugal filter devices from Millipore (Billerica, MA). The volumes of conditioned media loaded on gels were normalized to the protein concentrations of cell lysates. The fibronectin antibody was purchased from BD Transduction Laboratories, Inc. The Smad2 and phospho-Smad2 antibodies were obtained from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Lake Placid, NY). The uPA antibody (cat. no. 394) was obtained from American Diagnostica, Inc. The polyadenosine diphosphate ribose polymerase and Rho GDI (guanine nucleotide dissociation inhibitor) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The actin and FLAG antibodies were purchased from Sigma-Aldrich, Inc.

Transient Transfection and Luciferase Reporter Assay—Cells at 50–70% confluence on 12-well plates were co-transfected with 0.5 µg of a firefly luciferase promoter-reporter construct and 0.01 µg of the Renilla reniformis luciferase reference reporter construct, phRL-TK (Promega, Madison, WI) using Lipofectamine Plus reagents (Invitrogen). Four hours after transfection, cells were cultured back in regular media. Forty-eight hours after transfection, firefly and R. reniformis luciferase activities were measured using the Dual Luciferase Reporter Assay System kit (Promega) in an Optocomp II Luminometer (MGM Instruments, Inc., Hamden, CT). Normalized firefly luciferase activity was plotted as mean ± S.D. from three independent experiments. The phuPA-Luc reporter containing the nucleotide sequence –2345 to +30 of the human uPA promoter was kindly provided by Dr. Shuji Kojima (29). The p3TP-Lux reporter was a generous gift from Dr. Joan Massague (30).

Matrigel Invasion Assay—A modified Boyden chamber assay was performed using Transwells (12-µm pore size, 12 mm in diameter) from Costar (Cambridge, MA) and Matrigel (BD Biosciences). Each Transwell insert was first coated with 100 µl of 2.5 mg/ml Matrigel diluted in serum-free media for 1 h at 37 °C, and then 10 µl of Matrigel was added in the center of the Transwell 2 h before use. Cells were trypsinized, washed with serum-free media twice, re-suspended in 0.2% bovine serum albumin serum-free medium, seeded in Transwell inserts (150,000 cells/insert), and grown in the presence of 10% fetal bovine serum media in the lower chamber. After 16 h of incubation, Matrigel and cells remaining inside the inserts were removed with Q-tips, and the cells that had traversed to the reverse side of the inserts were rinsed with PBS, fixed in 4% formaldehyde for 30 min at room temperature, and stained with 1% crystal violet for 1 h to overnight at room temperature. Cells were counted under a light microscope (at 200x power), and invasive cell numbers were the averages of those from five areas on each insert. Each invasion assay was performed in triplicate and repeated three times.


Figure 1
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  		FIGURE 1.
The TGF-beta receptor kinase inhibitor, LY364947, inhibits TGF-beta signaling and MDA-MB-231 cell invasion. A, LY364947 inhibition of Smad2 phosphorylation induced by TGF-beta. Cells were treated with vehicle, LY364947, TGF-beta or in combination as indicated. 45 min later, cell lysates were collected and subjected to immunoblotting for total and phospho-Smad2. B, LY364947 inhibition of basal and TGF-beta-induced fibronectin expression. Cells were treated with vehicle, LY364947, TGF-beta, or in combination as indicated. 24 h later, lysates were collected and subjected to immunoblotting for fibronectin and beta-actin. C, LY364947 inhibition of basal and TGF-beta-induced promoter activation. Cells were co-transfected with the p3TP-Lux reporter construct and a reference reporter construct for 4 h as described under "Materials and Methods" and then treated with vehicle, LY364947, TGF-beta, or in combination as indicated. Luciferase activity was determined 48 h after treatments. D, LY364947 inhibition of MDA-MB-231 cell invasion. Cells were treated with LY364947 at the indicated concentrations overnight and then plated in Matrigel-coated Transwells in the presence of vehicle or increasing concentrations of LY364947 as indicated. 16 h later, cells on the reverse side of the Transwell membrane were stained and counted. Results represent the average from three independent experiments ± S.D. (*, p < 0.05; and **, p < 0.003 are derived from one-way ANOVA with a Bonferroni correction).

 
Adenovirus-mediated Overexpression of a Dominant-negative TGF-beta Type II Receptor (DNIIR)—To amplify adenoviruses, 293T cells cultured in 5% serum/Dulbecco's modified Eagle's medium at 80% confluence on P100 plates, were infected with adenoviruses in 1 ml of fresh 5% serum medium with rocking. After 3 h, 9 ml of 5% serum medium was added in each plate without removing infection medium. Three days after infection, cells were trypsinized, collected in 1 ml of 5% fetal bovine serum medium, and subjected to three freeze/thaw cycles at –20 °C/37 °C. Adenovirus-containing supernatant was obtained from the cell suspension by centrifugation at 15,000 x g for 20 min at 4 °C and stored at –80 °C. The relative values of multiplicity of infection (m.o.i.) of adenoviral suspension were determined by examining the cytopathic effect in 293T (adenovirus at m.o.i. values of 10–20 causes a total CPE in confluent 293T cells 3 days post infection). To perform adenoviral infection, 70–80% confluent cells were washed with PBS once, incubated with adenovirus-containing medium for 3 h, and then grown in regular serum media. Lysates and conditioned media were harvested 48–72 h post infection.

Cell Proliferation Assay—Cells were seeded in 96-well plates (50,000/well), and the relative viable cell numbers were determined by MTT assay using the CellTiter 96 Non-radioactive Cell Proliferation Assay kit (Promega), following the manufacturer's protocol. MTT hydrolysis was determined by measuring the absorbance at 570 nm using a plate reader.

Preparation of Plasma Membrane Fractions—Cells were collected into buffer containing 0.15 M NaCl, 20 mM HEPES, 2 mM CaCl2, 100 µg/ml leupeptin, 2.5 mg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride (pH 8.0) by scraping and then lysed by freeze/thaw (liquid N2/42 °C) cycles. Nuclei were isolated from the suspension of lysed cells by centrifugation at 500 x g for 20 min at 4 °C, washed three times, and re-suspended in radioimmune precipitation assay buffer. The nucleus-free supernatant was spun at 100,000 x g for 1 h at 4 °C. The resulting supernatant was compose of cytoplasmic fractions, and the pellets were subsequently washed three times with 3 ml of the cell resuspension buffer and dissolved in radioimmune precipitation assay buffer as membrane fractions.

MMP Zymography—Serum-free conditioned medium was mixed with 2x sample buffer (0.5 M Tris-HCl, pH 6.8, 5% SDS, 20% glycerol, and 1% bromphenol blue) and subject to SDS-PAGE using 10% SDS-gelatin (1 mg/ml final concentration) gels under a non-reducing condition. After electrophoresis, gels were soaked in washing buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 2.5% Triton X-100) for 1 h at room temperature to remove SDS and then in reaction buffer (50 mM Tris-HCl, pH 7.5, and 5 mM CaCl2, pH 8.0) overnight at 37 °C. Subsequently, gels were stained in staining buffer (0.15% Coomassie Blue R250 in 10% acetic acid and 30% methanol) and de-stained in the same staining solution without Coomassie Blue R250. Clear bands of pro- and active MMP-9 (92 and 84 kDa, respectively) were observed against the blue background of stained gels.


Figure 2
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  		FIGURE 2.
A dominant-negative TGF-beta type II receptor (DNIIR) suppresses TGF-beta signaling and MDA-MB-231 cell invasion. A, adenovirus-mediated expression of DNIIR-FLAG. Cells were infected with beta-galactosidase (Ad-beta-Gal) or DNIIR (Ad-DNIIR) adenoviruses at the indicated m.o.i. values. 48 h later, lysated cells were collected and subjected to immunoblotting for FLAG and beta-actin. B and C, DNIIR inhibited Smad2 phosphorylation and fibronectin expression induced by TGF-beta. 48 h after adenoviral infection, cells were treated with TGF-beta for 45 min and 24 h and then lysed for phospho-Smad2 (B) and fibronectin (C) immunoblotting, respectively. D, DNIIR inhibited basal and TGF-beta-induced promoter activation. 24 h after adenoviral infection, cells were transiently co-transfected with p3TP-Lux and a reference reporter construct for 4 h and then allowed to grow in the presence or absence of 5 ng/ml TGF-beta. 48 h after TGF-beta treatments, cells were lysed, and the luciferase activity in lysates was determined. E, suppression of cell invasion by DNIIR expression. 48 h after adenoviral infection, cells were plated in Matrigel-coated Transwells. 16 h later, cells on the reverse side of the Transwell membrane were stained and counted. (*, p < 0.05; **, p < 0.01; derived from one-way ANOVA with a Bonferroni correction.)

 
Northern Blot Analysis—Total RNA isolated with the TRIzol reagent was resolved on formaldehyde-agarose gels, transferred, and immobilized onto Hybond-N nylon membranes (Amersham Biosciences). Blots were blocked in the ULTRAHyb buffer (Ambion, Austin, TX) for 3 h at 65°C and then probed with 32P-labeled antisense Riboprobes (5 x 105 cpm/ml) in the ULTRAHyb buffer overnight at 68 °C. After several washes with low stringency (2x SSC/0.1% SDS) and high stringency (0.1x SSC/0.1% SDS) buffers at 68 °C, images were acquired by autoradiography using a PhosphorImager. To prepare Riboprobes, cDNA plasmids were linearized, purified, and then subject to in vitro transcription using the MAXIscript kit (Ambion) in the presence of 50 µCi of [{alpha}-32P]UTP (800 Ci/mmol) for 1 h at 37 °C. Unincorporated nucleotides were removed using Nuc-Away columns (Ambion).

Nuclear Run-on Assay—Cell were collected, washed twice with PBS, and then re-suspended in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 1 mM dithiothreitol). Nonidet P-40 was then added to a final concentration of 0.2–0.5%, depending on cell types. After a 5-min incubation on ice, nuclei were pelleted at 500 x g for 5 min, washed once with nuclear freezing buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 1 mM dithiothreitol), and re-suspended in fresh nuclear freezing buffer. In vitro run-on transcription was performed using 2 x 107 nuclei in 150 µl of reaction buffer (5 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2, 150 mM KCl, 1 mM of ATP, CTP, or GTP, 150 µCi of [{alpha}-32P]UTP (800 Ci/mmol), 80 units of RNasin, and 2.5 mM dithiothreitol) for 30 min at 30 °C. Transcription was terminated by adding 350 µl of deoxyribonuclease I solution (20 mM Tris-HCl, pH 7.4, 10 mM CaCl2, and 300 units of RNase-free DNase I) and a 30-min incubation at 37 °C. Next, proteins were digested by adding 50 µl of proteinase K solution (1% SDS, 5 mM EDTA, 1 mM Tris-HCl, pH 7.4, and 300 µg/ml proteinase K) and a 30-min incubation at 50 °C. The 32P-labeled RNAs were phenol/chloroform purified and precipitated in 10% trichloroacetic acid plus 20 µg of yeast tRNA. After centrifugation at 15,000 x g for 1 h, the RNAs were re-suspended in RNase-free H2O, denatured for 10 min at 65 °C, and then chilled on ice. The radiolabeled RNAs were hybridized to cDNAs pre-immobilized on membranes in hybridization buffer (50% formamide, 5x SSC, 5 mM EDTA, 5x Denhardt's solution, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA) for 48 h at 42 °C. Next, the membranes were washed several times in 2x SSC (1x SSC = 0.15 M NaCl and 12.5 mM sodium citrate, pH 7) and then in 2x SSC plus 10 µg/ml RNase A for another 30 min or not depending on intensity of background. Signals were acquired and quantified by a PhosphorImager. To immobilize cDNAs to nitrocellulose membranes, 1 µg of linearized plasmid was denatured in 0.2 M NaOH for 30 min at room temperature and then neutralized with 10 volumes of 6x SSC. The DNAs were applied onto nitrocellulose membranes using a slot blot apparatus and immobilized by UV cross-linking.

RNA Interference—To perform Smad4 silencing, 50% confluent cells were transfected with 50–200 pM of a pool of four Smad4 or scrambled siRNAs (Dharmacon, Lafayette, CO) using Oligofectamine (Invitrogen) according to manufacturer's guideline. Conditioned media (48–72 h post transfection) and protein lysates (72 h post transfection) were harvested and subjected to immunoblotting for Smad4 and uPA. To determine uPA mRNA stability under a Smad4-silencing condition, cells were first transfected with 100 nM Smad4 siRNA for 6 h and grew in serum media overnight, and then uPA mRNA stability was determined following treatment of TGF-beta overnight.

Statistical Analysis—p values for multiple comparison tests were derived by an analysis of variance (ANOVA) with a Bonferroni correction.


Figure 3
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  		FIGURE 3.
Inhibition of TGF-beta signaling decreases uPA but not MMP-9 protein levels in MDA-MB-231 conditioned media. A, down-regulation of uPA secretion by LY364947. Cells were treated with LY364947 at the indicated concentrations for 24 h. Conditioned media, cell lysates, and total RNAs were collected and subjected to immunoblotting for uPA and beta-actin and Northern blotting for uPA and cyclophilin. B, time course of LY364947 inhibition on uPA secretion. Cells were treated with 5 µM LY364947 for 2, 4, 8, 16, and 24 h. Conditioned media were collected, concentrated (early time point media), and subjected to immunoblotting for uPA. C, the effect of LY364947 on uPA protein levels on cellular membrane. Cells were treated with 5 µM LY364947 or 5 ng/ml TGF-beta for 24 h. Membrane, cytoplasmic, and nuclear fractions were isolated as described under "Materials and Methods" and subjected to immunoblotting for uPA, polyadenosine diphosphate ribose polymerase (a nuclear marker), Rho guanine nucleotide dissociation inhibitor (GDI) (a cytoplasmic marker), and beta-actin. D, down-regulation of uPA secretion by the dominant-negative TGF-beta type II receptor (DNIIR). Cells were infected with beta-galactosidase (Ad-beta-Gal) or DNIIR (Ad-DNIIR) adenoviruses at the indicated m.o.i. values. 48 h later, cells were cultured in fresh medium and allowed to grow for 16 h. Conditioned media, protein lysates, and total RNAs were harvested and subjected to immunoblotting for uPA and beta-actin and Northern blotting for uPA and cyclophilin. E, the effect of LY364947 on pro-MMP-9 and active MMP-9 protein levels. Cells were treated with LY364947 at the indicated concentrations for 24 h. Condition media were collected and subjected to gelatin zymography.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Autocrine TGF-beta Signaling Contributes to MDA-MB-231 Cell Invasion—The metastatic MDA-MB-231 breast cancer cells secrete active TGF-beta (22, 23) and are TGF-beta-responsive (24). The importance of autocrine TGF-beta signaling in regulation of MDA-MB-231 cell invasiveness was assessed by Matrigel invasion assays following abrogation of autocrine TGF-beta signaling using the LY364947 compound (27, 28), a kinase inhibitor of TGF-beta type I receptor (TbetaRI) by functioning as a potent ATP competitive inhibitor.

The inhibitory effect of LY364947 on TGF-beta signaling was first validated by examining Smad2 phosphorylation. Basal levels of Smad2 phosphorylation were undetectable by immunoblotting. However, LY364947 abolished Smad2 phosphorylation induced by exogenous TGF-beta without altering total Smad2 protein levels (Fig. 1A). Expression of fibronectin is induced by TGF-beta through a Smad-independent pathway (31). LY364947 decreased both basal and exogenous TGF-beta-induced fibronectin expression (Fig. 1B). In addition, we evaluated the effect of LY364947 on TGF-beta-induced promoter activation by reporter assays using p3TP-Lux, a luciferase reporter construct highly responsive to TGF-beta (30) and observed that LY364947 significantly inhibited both basal and exogenous TGF-beta-induced promoter activation (Fig. 1C). To investigate whether autocrine TGF-beta has a role in regulation of MDA-MB-231 invasiveness, Matrigel invasion assays were performed with or without LY364947 treatment. LY364947 inhibited cell invasion in a dose-dependent manner (Fig. 1D). These data suggest that autocrine TGF-beta plays a role in basal invasive growth of MDA-MB-231 cells.

A Dominant-negative TGF-beta Type II Receptor Suppresses Autocrine TGF-beta Signaling and Cell Invasion—To substantiate the results obtained using the TGF-beta receptor kinase inhibitor, we assessed invasiveness of MDA-MB231 cells following suppression of autocrine TGF-beta signaling with a dominant-negative TGF-beta type II receptor (TbetaRII). TbetaRII is the receptor responsible for ligand binding and for activation of TGF-beta type I receptor through its kinase activity. TbetaRII devoid of the kinase domain (DNIIR) acts as a dominant-negative mutant by competing with wild-type receptors for TGF-beta ligands (32). Expression of FLAG-tagged dominant-negative DNIIR was achieved using an adenoviral vector and was confirmed by immunoblotting for FLAG (Fig. 2A). DNIIR expression was increased with increasing amounts of adenovirus, whereas no DNIIR was detected in parental or the beta-galactosidase adenovirus-infected cells. Expression of DNIIR inhibited TGF-beta-stimulated Smad2 activation (Fig. 2B) and decreased both basal and exogenous TGF-beta-induced fibronectin expression (Fig. 2C). DNIIR also suppressed basal and exogenous TGF-beta-stimulated p3TP-Lux promoter activation (Fig. 2D), demonstrating the inhibitory effects of DNIIR on TGF-beta signaling. As expected, DNIIR expression significantly decreased MDA-MB-231 cell invasion (Fig. 2E). This effect is not a result of inhibition of cell proliferation as determined by MTT assay (data not shown). Thus, consistent with the results using the pharmacological inhibitor, inhibition of autocrine TGF-beta signaling by the dominant-negative TGF-beta type II receptor (DNIIR) down-regulated MDA-MB-231 invasiveness.


Figure 4
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  		FIGURE 4.
TGF-beta regulates uPA production and cell invasion of Panc-1 pancreatic cancer cells. A, paracrine/exogenous and autocrine TGF-beta regulation of uPA production. Cells were treated with TGF-beta or LY364947 at the indicated concentrations for 48 h. Conditioned media and total RNAs were collected and subjected to immunoblotting for uPA and beta-actin and Northern blotting for uPA and cyclophilin. B, cells were treated with TGF-beta or LY364947 at the indicated concentrations for 48 h and then subjected to invasion assays. (*, p < 0.03; derived from one-way ANOVA with a Bonferroni correction.)

 
Disruption of Autocrine TGF-beta Signaling Suppresses uPA Secretion—The MDA-MB-231 cells secrete uPA (20). To address whether autocrine TGF-beta can modulate uPA expression, uPA levels were determined after blockade of autocrine TGF-beta signaling using LY364947. Immunoblotting for uPA was conducted under non-reducing conditions, which detect both uPA and complexes of uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1) (33). The low motility bands with molecular masses around 100 kDa represent uPA·PAI-1 complexes (uPA ~55 kDa and PAI-1 ~52 kDa). Our results show that LY364947 decreased uPA secretion in a dose-dependent manner (Fig. 3A) and that the decreased level of free uPA in the conditioned medium did not result from increased uPA/PAI-1 association and depletion of uPA. The kinetics of the LY364947 effect was determined by a time-course experiment from 2 to 24 h after treatment with 5 µM LY364947. The result (Fig. 3B) shows that uPA secretion was inhibited starting 8 h after treatment. MDA-MB-231 cells constitutively express uPA receptor mRNA and exhibit detectable membrane-associated uPA (20). To determine whether the decreased level of released uPA after LY364947 treatment was the result of increased uPA receptor expression and increased uPA binding to uPA receptor, levels of membrane-bound uPA were determined after LY364947 treatment. The purity of the membrane fractions was confirmed by the absence of nuclear and cytoplasmic proteins, polyadenosine diphosphate ribose polymerase, and Rho GDI (guanine nucleotide dissociation inhibitor), respectively (Fig. 3C). Our results show that neither exogenous TGF-beta treatment nor LY364947 treatment altered membrane uPA levels (Fig. 3C). Inhibition of autocrine TGF-beta signaling by expression of DNIIR also suppressed uPA secretion in a dose-dependent fashion (Fig. 3D). In contrast, beta-galactosidase adenovirus infection did not alter uPA secretion, further supporting that inhibition of autocrine TGF-beta signaling suppresses uPA secretion. Of note, expression of DNIIR was not detectable at an m.o.i. of 10 by immunoblotting (Fig. 2A); however, this low level of DNIIR expression was sufficient to decrease uPA secretion (Fig. 3D). These data demonstrate that disruption of autocrine TGF-beta signaling inhibited uPA secretion.

Steady-state levels of uPA mRNA did not change with either LY364947 treatment or DNIIR expression (Fig. 3, A and D). Further, whereas uPA release into the medium was decreased, the intracellular uPA were increased after blockade of autocrine TGF-beta signaling (Fig. 3, A and D), suggesting that autocrine TGF-beta stimulates uPA secretion and that inhibition of autocrine TGF-beta signaling reduces the level of secretion without impairing uPA production, thus leading to intracellular accumulation of uPA. We also examined uPA production in response to TGF-beta in the Panc-1 human pancreatic cancer cell line and found that TGF-beta treatment stimulated overall uPA production (free uPA plus uPA in complex with PAI-1), whereas LY364947 decreased overall uPA production with no change in uPA mRNA levels (Fig. 4A). In this cell line, total uPA levels also correlate with invasive activity (Fig. 4B). These data demonstrate that the TGF-beta-regulated uPA secretion is not limited to a single cell line, and the Panc-1 produced TGF-beta also regulated uPA production and invasion in an autocrine manner.

Inhibition of Autocrine TGF-beta Signaling Does Not Affect MMP-9 Protein Levels and Activity—MDA-MB-231 cells express the MMP-9 protein (20) that is particularly important among at least 19 MMP proteins identified to date in tumor invasion and metastasis due to its ability to degrade the basement membrane component, type IV collagen. We determined MMP-9 protein levels and activity following abrogation of autocrine TGF-beta signaling with LY364947. Gelatin zymography shows no change in both pro-(92 kDa) and active MMP-9 (84 kDa) protein levels after LY364947 treatment (Fig. 3E). These results indicate that autocrine TGF-beta does not regulate MMP-9 activity or protein expression in the MDA-MB-231 cells. Consistent with previous reports (20), we did not detect MMP-2 (72 kDa) expression in MDA-MB-231 cells.

Inhibition of Basal uPA Activity Impairs MDA-MB-231 Cell Invasion—Inhibition of autocrine TGF-beta signaling resulted in decreased invasiveness and uPA secretion as described above. We next investigated a relationship between uPA activity and MDA-MB-231 cell invasiveness. Inhibition of uPA activity using an anti-catalytic uPA blocking antibody attenuated cell invasion by ~70% as compared with no treatment or IgG treatment in Matrigel invasion assays (Fig. 5A). These data suggest a correlation between uPA activity and MDA-MB-231 cell invasiveness. Parallel MTT assays suggest that the decreased cell invasion was not due to inhibition of cell proliferation (Fig. 5B). To test the hypothesis that uPA is the molecule that mediates the autocrine TGF-beta pro-invasive effect, we examined whether addition of uPA in the medium can reverse the inhibitory effect of LY364947 on cell invasion. Our result shows LY364947 failed to decrease cell invasion in the presence of recombinant uPA (Fig. 5C), suggesting that uPA is the cellular molecule that mediates TGF-beta-regulated cell invasion. Interestingly, addition of recombinant human PAI-1, an inhibitor of uPA, did not decrease basal cell invasion. As PAI-1 has been previously shown to have a positive role in cell attachment and invasion (34), and concomitant elevation of uPA and PAI-1 has been observed in cancer and linked to poor outcome (35), our data support the hypothesis that invasive potential can be up-regulated by increased uPA expression in conditions with elevated PAI-1 expression.


Figure 5
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  		FIGURE 5.
A blocking antibody of uPA activity decreases tumor cell invasiveness. Cells were plated and grown in Matrigel-coated Transwells (A) or 96-well plates (B) in the presence or absence of 50 µg/ml normal IgG or an uPA blocking antibody. 16 h later, invasive and viable cell numbers were determined by Matrigel invasion and MTT assays, respectively. C, cells were pre-treated with LY364947 overnight or not and then grown in Matrigel-coated Transwells in the presence of 10 µM LY364947, 200 nM purified human uPA, 250 nM recombinant human PAI-1 (r-PAI-1), or in combination. 16 h later, invasive cell numbers were determined. (*, p < 0.05; derived from one-way ANOVA with a Bonferroni correction.)

 


Figure 6
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  		FIGURE 6.
The Smad pathway is required for uPA secretion. A, LY364947 did not alter the activation status of ERK1/2, Akt, p38, and Src proteins. Cells were treated with 0, 1, and 10 µM LY364947 for 24 h and then lysed. Immunoblotting of the lysates was performed using the antibodies against total and phospho-ERK1/2, Akt, p38, and Src proteins. B, Smad4 silencing decreased uPA secretion. Cells were transiently transfected with Smad4 or scrambled control siRNAs at the indicated concentrations for 24 h. Cells were then allowed to grow in fresh medium for another 24 h. Conditioned media and lysates were harvested and subjected to immunoblotting for uPA, Smad2, Smad4, and beta-actin. C, forced expression of Smad4 blocked the inhibitory effect of Smad4 silencing on uPA secretion. Cells were transfected with Smad4 siRNAs. On the next day, cells were infected with beta-galactosidase or Smad4 adenoviruses at the indicated m.o.i. 48 h after viral infection, conditioned media and lysates were harvested and subjected to immunoblotting for uPA, Smad4, and beta-actin.

 
Smad4 RNA Interference Decreases uPA Secretion—TGF-beta receptors transduce signals through both Smad and non-Smad pathways, including the extracellular signal-regulated kinases (ERK1/2) (7, 8), the p38 mitogen-activated protein kinase (9, 10), the phosphatidylinositol 3-kinase (PI3K) (12), and the Src (11) pathways. Whether autocrine TGF-beta regulates these Smad-independent pathways was evaluated by immunoblotting. LY364947 treatment (up to 10 µM) did not significantly alter activation status of ERK1/2, p38, Akt, and Src proteins (Fig. 6A). In contrast, as little as 0.1µM LY364947 suppressed uPA secretion (Fig. 3A), implicating that these pathways are probably not involved in autocrine TGF-beta regulation of uPA secretion. We hypothesized that the Smad pathway mediates the autocrine TGF-beta effect on uPA secretion. In this case, down-regulation of the common Smad mediator, Smad4, should suppress uPA release in response to autocrine TGF-beta signaling. Smad4 silencing was achieved using a mixture of four Smad4 siRNAs. Immunoblotting results confirmed down-regulation of Smad4 protein expression in Smad4 siRNA-transfected cells but not in parental or scrambled control siRNA-transfected cells (Fig. 6B). Smad2 protein expression was unaffected by the Smad4 siRNA transfection, further confirming specific silencing of Smad4 protein expression by the Smad4 siRNAs. Interestingly, uPA secretion was also decreased by inhibition of Smad4 protein expression (Fig. 6B), suggesting that the Smad pathway is required for the regulation of uPA secretion by autocrine TGF-beta. To further confirm the Smad4 role in uPA secretion, we tested whether forced expression of Smad4 can rescue the inhibitory effect of Smad4 silencing on uPA secretion. Fig. 6C shows that adenovirus-mediated overexpression of Smad4 prevented the inhibited uPA secretion induced by Smad4 silencing.

Paracrine TGF-beta Increases uPA mRNA Levels through RNA Stabilization—Paracrine/exogenous TGF-beta was previously shown to stimulate uPA expression and increased MDA-MB-231 cell invasiveness (20). We further examined uPA regulation by exogenous TGF-beta. Unlike autocrine TGF-beta, exogenous TGF-beta increased both uPA mRNA and protein levels in a dose-dependent manner (Fig. 7A). A time-course study shows that uPA mRNA was induced by TGF-beta in a time-dependent fashion to near maximal levels by 16 h after treatment, and the level was sustained for at least 48 h (Fig. 7B). Increased mRNA expression is due to either increased transcription or increased RNA stability. The transcription of uPA in response to exogenous TGF-beta was determined by a nuclear run-on assay. Exogenous TGF-beta failed to enhance uPA transcription (Fig. 8A) but strongly stimulated transcription of PAI-1, a TGF-beta target gene that has been shown to be transcriptionally activated by TGF-beta (36). Consistent with the nuclear run-on result, transient expression assays using a uPA-promoter-reporter construct containing the nucleotide –2345 to +30 region of the human uPA promoter (phuPA-Luc) show very little changes in uPA promoter activity after TGF-beta treatment (Fig. 8B). In contrast, uPA promoter activity was significantly induced by phorbol 12-myristate 13-acetate, a known inducer of activation of the human uPA promoter (37) (Fig. 8B). Next, the stability of uPA mRNA in the presence and absence of exogenous TGF-beta was determined by examining uPA mRNA levels at various time points after blocking transcription with the RNA polymerase II-specific inhibitor DRB (5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole). Quantitation of the Northern blotting results (Fig. 8C) shows that by 8 h after DRB treatment, uPA mRNA levels were decreased by 50% in untreated cells. In contrast, uPA mRNA levels were only decreased by 25% in TGF-beta-treated cells, suggesting that exogenous TGF-beta enhances the stability of uPA mRNA. Thus, exogenous TGF-beta increased uPA mRNA levels through mRNA stabilization in MDA-MB-231 cells.


Figure 7
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  		FIGURE 7.
Paracrine TGF-beta increases uPA mRNA levels and protein secretion in a dose- and time-dependent manner. A and B, cells were treated with increasing concentrations (0.1–10 ng/ml) of TGF-beta for 24 h (A) or 5 ng/ml TGF-beta for various intervals (B) as indicated. Conditioned media, lysates, and total RNAs were then harvested and subjected to immunoblotting for uPA and Northern blotting for uPA and cyclophilin, respectively.

 


Figure 8
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  		FIGURE 8.
Paracrine TGF-beta stabilizes uPA mRNA. A, TGF-beta treatment did not stimulate uPA transcription as determined by nuclear run-on assays. Nuclei were harvested from cells treated with TGF-beta for 0, 3, and 24 h and subjected to a nuclear run-on assay as descried under "Materials and Methods." Signals were visualized and quantified by a PhosphorImager and plotted as -fold changes. B, TGF-beta treatment did not increase uPA promoter activity. Cells were co-transfected with phuPA-Luc and a reference reporter construct for 24 h and then treated with TGF-beta or phorbol 12-myristate 13-acetate. 48 h later, cells were lysed and the luciferase activity in lysates was determined. C, stabilization of uPA mRNA by paracrine/exogenous TGF-beta. Cells were treated with vehicle or 5 ng/ml TGF-beta for 16 h and then with 20 µg/ml of DRB plus vehicle or TGF-beta. Total RNAs were isolated at the indicated time points after DRB treatment and subjected to Northern blotting for uPA and cyclophilin. Cyclophilin-normalized uPA mRNA levels of the results were plotted as percentages of the uPA level at the 0 h time point. (*, p < 0.05; **, p < 0.001; derived from one-way ANOVA with a Bonferroni correction.)

 
Smad4 Is Required for Paracrine TGF-beta-mediated Stabilization of uPA mRNA—The engagement of the Smad pathway in the paracrine/exogenous TGF-beta-induced effect on uPA stability was determined. We first examined steady-state levels of uPA mRNA following TGF-beta treatment in Smad4 knock-down cells. Suppressed Smad4 protein expression was first confirmed following Smad4 siRNA transfection with or without TGF-beta treatment (Fig. 9A). Interestingly, uPA mRNA induction by TGF-beta was not observed in the Smad4 siRNA-transfected cells, suggesting that the uPA mRNA-stabilizing effect of exogenous TGF-beta is lost in the absence of Smad4. We next examined the stability of uPA mRNA following TGF-beta treatment in control or Smad4 siRNA-transfected cells and observed that TGF-beta failed to stabilize uPA mRNA in Smad4 siRNA-transfected cells (Fig. 9, A and B). These data suggest that the Smad pathway is required for uPA mRNA stabilization induced by exogenous TGF-beta. There was not complete loss of the TGF-beta effect on uPA mRNA stability in Smad4 siRNA-transfected cells, presumably attributed to undetected residual expression of Smad4 protein under the Smad4 silencing condition.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGF-beta is a potent inhibitor of epithelial cell growth through inhibition of proliferation and induction of apoptosis (38, 39) and is an important tumor suppressor (40). The tumor suppressor role of TGF-beta is evident in that mice heterozygous for deletion of the TGF-beta gene with expression of 10–30% of TGF-beta levels of wild-type animals, developed an increased number of chemically induced tumors than did wild-type littermates (40). However, escape from the growth inhibitory effects of TGF-beta occurs frequently in cancer through numerous mechanisms. Moreover, TGF-beta displays tumor promoting effects in late-staged tumors. TGF-beta has been shown to facilitate tumor progression by inducing epithelial to mesenchymal transition (41, 42), ECM degradation (20), and cyclooxygenase-2 expression (43) or by inhibiting antitumor immune responses (44).


Figure 9
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  		FIGURE 9.
Smad4 is required for paracrine TGF-beta-mediated stabilization of uPA mRNA. A, Smad4 RNA silencing inhibited uPA mRNA expression induced by TGF-beta treatment. MDA-MB-231 cells were transfected with 200 nM siRNA as described under "Material and Methods." 24 h after transfection, cells were treated with 5 ng/ml TGF-beta in 1% serum media overnight. Cell lysates and total RNA were then harvested for Smad4 and beta-actin immunoblotting and for uPA and cyclophilin Northern blotting, respectively. The graph shows the quantitation of cyclophilin-normalized uPA mRNA levels. B, MDA-MB-231 cells were transfected with 200 nM siRNA. On the next day, cells were treated with 5 ng/ml TGF-beta in 1% serum media. 24 h after TGF-beta treatment, cells were treated with DRB plus vehicle or TGF-beta, and uPA mRNA levels were determined at 0, 2, 4, 6, and 8 h post DRB treatment. The graphs show quantitation of cyclophilin-normalized uPA mRNA levels.

 
Disruption of tumor autocrine TGF-beta signaling has been found to delay tumor growth and inhibit metastases (13, 45). However, the molecular mechanisms underlying the tumorigenic effects of autocrine TGF-beta remain unclear. The present study demonstrates that autocrine TGF-beta regulates both cell invasiveness and uPA secretion. Inhibition of uPA activity decreases tumor cell invasion to the same extent as does the inhibition of autocrine TGF-beta signaling. Interestingly, although autocrine TGF-beta regulates uPA availability via effects on protein secretion, paracrine/exogenous TGF-beta further increases uPA availability by increasing uPA mRNA through RNA stabilization. Our data suggest two distinct levels of regulation of uPA in response to different magnitudes of stimulation by TGF-beta. The amount of active TGF-beta in MDA-MB-231 cell-conditioned media has been quantitated at ~0.25 ng/ml (22, 23, 46), whereas at least 10-fold higher concentrations were required to increase uPA mRNA levels (see Fig. 6A).

In the secretory pathway, proteins are transported between intracellular compartments via membranous vesicles that the process involves vesicle formation, motility, and docking, and membrane remodeling and fusion (47, 48). Numerous proteins have been implicated in regulation of vesicle trafficking during protein secretion. For example, members of Ypt/Rabs proteins of the Ras GTPase superfamily have been characterized to be key regulators of protein transport (49–51). A negative role for Rab3, a member of the Ypt/Rab family, in protein secretion has been previously shown by a number of studies (reviewed in Ref. 52). This function of Rab3 suggests that depletion of Rab3 may lead to enhanced protein secretion. Recently, one of the biological functions of TGF-beta has been shown to be facilit at ingubiquitin-dependent degradation of protein in a Smad-dependent manner (53). These observations raise the possibility that TGF-beta signaling may modulate uPA protein secretion by depleting negative regulators of protein secretion through facilitated protein degradation.

Most of TGF-beta activities involve modulation of gene transcription. However, TGF-beta has been shown to substantially increase stability of mRNAs such as {alpha}1(I) collagen mRNA in human hepatic stellate cells (54) and elastin mRNA in human fetal lung fibroblasts (55). In both cases, activation of the p38 MAPK signaling pathway is required for the TGF-beta effect. Interestingly, expression of inhibitory Smad7, an inhibitor of TGF-beta signaling but not Smad6, an inhibitor of bone morphogenetic protein action blocked TGF-beta signaling and dramatically diminished the TGF-beta-stabilizing effect on elastin mRNA (55). Theses results suggests the involvement of both Smad and p38 MAPK pathways in the TGF-beta-mediated stabilization of elastin mRNA. In our study, exogenous TGF-beta stabilized uPA mRNA in a Smad4-dependent manner. Exogenous TGF-beta also induced activation/phosphorylation of p38 MAPK (data not shown) in the same cell context. Therefore, it is conceivable that the p38 signaling pathway may participate in the process of TGF-beta regulation of uPA mRNA stability in conjunction with the Smad pathway.

Stabilization of mRNA involves the binding of RNA proteins to certain cis-elements of mRNAs (56). Adenylate-uridylaterich elements are important regulatory cis-elements present in the untranslated regions of short-lived mRNAs such as protooncogenes, cyclooxygenase-2 (57), and c-fos (58) mRNAs. TGF-beta and Ras have been shown to synergistically stabilize the COX-2 mRNA through an adenylate-uridylate-rich element in the proximal 3'-untranslated regions (57). Given that MDA-MB-231 cells possess an activating Ki-Ras mutation (59) and that adenylate-uridylate-rich elements are present in the 3'-untranslated region of the uPA mRNA (60), it is possible that TGF-beta may cooperate with the active Ras to regulate the stability of uPA mRNA through a similar mechanism, but confirmation of this awaits further investigation.

Cells release uPA as a single-chain zymogen. The low level of intrinsic proteolytic activity of pro-uPA (61) can convert the plasminogen in tumor microenvironments or Matrigel (62) to plasmin, which in turn activates pro-uPA. This pro-uPA activation by plasmin and activation of plasminogen by uPA in a cyclic fashion promotes degradation of the ECM or the basement membrane and facilitates cell invasion. In addition, plasmin can potentially activate MMPs (15, 63, 64) thereby promoting ECM degradation and tumor cell invasion (65). However, we did not find evidence for up-regulation of MMP-9 activity or expression by TGF-beta in the MDA-MB-231 cells under the conditions studied.

TGF-beta is released from cells mostly in a latent, inactive form via a constitutive secretion pathway (66). Despite the predominance of latent TGF-beta in conditioned media in general, MDA-MB-231 cells express detectable active TGF-beta (46). It is interesting that uPA can proteolytically activate latent TGF-beta (67). Therefore, TGF-beta regulation of uPA production may be a positive feedback loop for activation of latent TGF-beta (Fig. 8), and this relationship can be a cycle in cancer progression because TGF-beta is overexpressed in both malignant breast tumors and surrounding stroma (68, 69), and uPA expression is increased in human breast carcinomas and bone metastases (70). It will be of interest to determine whether the basal level of uPA secretion contributes to the availability of active TGF-beta and autocrine TGF-beta signaling.

The plasminogen activator inhibitor (PAI-1) is a TGF-beta target gene and a strong inhibitor of uPA. The present study demonstrates that TGF-beta increases levels of both uPA and PAI-1 in the MDA-MB-231 conditioned media. Interestingly, despite the fact that PAI-1 inhibits uPA activity, concomitant elevation of uPA and PAI-1 has been observed in breast cancer and is associated with poor outcome (35), suggesting that tumor progression may occur in the presence of high levels of PAI-1. TGF-beta has been shown to stimulate attachment and invasion through up-regulation of PAI-1 (34), and these effects may contribute to cell invasion. Our findings suggest that, although PAI-1 is induced by TGF-beta, this induction is not sufficient to prevent the invasion promoting effect of uPA.

In summary, autocrine TGF-beta regulates cell invasiveness through maintaining uPA levels by facilitated protein secretion, whereas paracrine/exogenous TGF-beta further increases invasiveness through stimulated uPA expression by RNA stabilization. The Smad pathway appears to be required for the distinct levels of regulation of uPA in response to different magnitudes of TGF-beta stimulation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health (NIH) Grants DK52334 and CA69457 (to R. D. B.), the Vanderbilt-Ingram Cancer Center Support Grant P30 CA68485, Gastrointestinal Cancer SPORE Grant P50 CA95103, NIH Grant RO1 CA95195 (to P. K. D.), and NIH Grant RO1 CA093651 (to B. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Section of Surgical Sciences, Vanderbilt University Medical Center, 1161 21st Ave. South, D-4316 Medical Center North, Nashville, TN 37232-2730. Tel.: 615-322-2363; Fax: 615-343-5365; E-mail: daniel.beauchamp{at}vanderbilt.edu.

2 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TGF-beta, transforming growth factor-beta;TbetaRI, -II, transmembrane types I and II; ERK, extracellular signal-regulated kinase; DNIIR, dominant-negative type II receptor; uPA, urokinase plasminogen activator; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Natasha Dean and Cindy Lynn Kanies for their careful proofreading of the manuscript.



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